Treatment of microbial infections

ABSTRACT

The present application relates to an anti-microbial system for use in the treatment of microbial infections or control of microbial contamination, which avoids the use of antibiotics. Such infections include mastitis, tuberculosis, cystic fibrosis and the contamination that may result from biofilm formation on medical devices.

FIELD OF THE INVENTION

The present application relates to an anti-microbial composition for usein the treatment of microbial infections. In particular, the compositionmay be used to treat bacterial infections, or for control of bacterialcontamination, which avoids the use of antibiotics. Such infectionsinclude mastitis, tuberculosis, cystic fibrosis and other lunginfections, and the contamination that may result from biofilm formationon surfaces such as on medical devices. However, the composition is alsosuitable for the treatment of viral, yeast or fungal infections or forthe control of contamination by such organisms.

BACKGROUND TO THE INVENTION

Mastitis is a persistent inflammatory condition of the udder of cows andother milk-producing animals. It is one of the most common diseases indairy cows in the United States and is also the most costly to the dairyindustry. Mastitis occurs when white blood cells are released into themammary gland, usually in response to invasion of bacteria in the teatcanal. Milk from cows with mastitis has a higher somatic cell count, andthe higher the somatic cell count, the lower the quality of the milk.

Normal treatment for mastitis is with antibiotics, but milk fromantibiotic treated cows is not marketable, until the drug has left thecows' system. The antibiotics used may be systemic and injected into thebody, or they may be forced into the teat through the teat canal byintra-mammary infusion. Mastitis can be clinical, whereby visible signsof infection are noted or sub-clinical, where the presence of infectionis noted only by an increase in somatic count in the resulting milk. Insome clinical situations, cows are often left untreated, though revenueis lost to the dairy farmer through a reduction in the amount of moneypaid for the milk, which occur where there is an elevated somatic cellcount in the milk.

There are a number of other uses of the anti-microbial composition ofthe present invention. These include infections of the mammalian lung.Cystic fibrosis and tuberculosis are two diseases that are, at present,extremely difficult to treat. Tuberculosis symptoms are caused byinfection in the lungs and require long-term antibiotic treatment.Cystic fibrosis (CF) is a condition wherein the sufferer cannot regulatethe transfer of chloride ions across their membranes, particularly inthe lungs. The condition invariably results in numerous, chronic, lunginfections. Antibiotic treatment for either condition can lead toserious drug resistance, minimising their effectivness. At present,antibiotics are delivered through the blood stream intra-venously, or byoral suspension/tablets, or by inhalation. Drug delivery is a bigproblem for CF sufferers as the antibiotic cannot efficiently transversethe lung membrane to where it is required. This leads to problemswherein resistance to the drug, through the introduction ofsub-inhibitory concentrations, may become a serious issue. This makesany further treatment with the drug obsolete.

Burns patients, or patients with open wounds, are extremely susceptibleto bacterial infections, notably those due to Staphlycoccal species orPseudomonad species of bacteria. Treatment of such infections willinvariably be by a regimen of antibiotics, either oral orintra-venously. These may be given prophalactically, or when infectionis apparent. Such use of antibiotics will often lead to resistance tothe drug and an ineffective treatment outcome. The researchers envisiona new method of treating burns patients with the present technology.

In addition, large numbers of antibiotic treatments each year are due tothe result of medical devices that have become infected whilst in use bya patient. A large number of organisms are responsible for suchinfections, including both Gram-positive and Gram-negative bacteria.Infections, on such items as urinary or intra-venous catheters, areoften the result of the non-sterile installation of such devices. Overthe course of a number of days, any bacterial cells present on thesurface of the device will proliferate, leading to the production ofbiofilms. Such biofilms are extemely difficult to treat withanitbiotics, due to the poor transfer of the drug across to the innercells of the biofilm mass, leading often to even greater levels oftolerance of the biofilms to the antibiotic. Infection of the medicaldevice will often require its removal and replacement, to the discomfortof the patient. Although the infection will often be noted a number ofdays after installation of the medical device, it will be typicallyincurred as the result of bacteria being present very early in theinstallation.

It is generally known that a bacteriostatic effect is caused by thereaction between hydrogen peroxide and thiocyanate, catalysed bylactoperoxidase—a process referred to as the Lactoperoxidase (LP)system. In certain instances, the source of peroxide is a reactionbetween glucose and glucose oxidase, which results in the production ofgluconic acid and peroxide. This process is used during the transport ofmilk. Antibacterial treatments for the control of infections have beenproposed, based on the LP system. For example, PCT ApplicationWO2008/04128 discloses a preparation with an antimicrobial andimmuno-stimulatory effect, which comprises an oxidoreductase enzyme, anappropriate substrate for that enzyme to produce hydrogen peroxide, andendogenous hydrogen peroxide preparations. The preparation produces2-stage hydrogen peroxide release; the endogenous form of peroxideensuring that there is instantly available hydrogen peroxide and furtherhydrogen peroxide is produced by the oxidoreductase enzyme.

U.S. Pat. No. 6,312,687 describes a stabilised aqueous enzymeconcentration comprising lactoperoxidase, glucose oxidase, an alkalinemetal halide salt and a buffering agent, for use as an antimicrobialcomposition. U.S. Pat. No. 5,607,681 describes antimicrobialcompositions, which comprise iodide anions and thiocyanate anionstogether with D-glucose and either glucose oxidase or glucose oxidasetogether with and at least one antioxidant. The composition mayadditionally comprise lactoperoxidase.

The proposed basis for the bacteriostatic effect of the LP system inmilk is based on thiocyanate and a source of hydrogen peroxide. Thethiocyanate ion is oxidised in the presence of hydrogen peroxide bylactoperoxidase, to produce hypothiocyanous acid. In certainembodiments, the peroxide ion is produced from glucose by the action ofglucose oxidase rather than by using a solution of hydrogen peroxide orits release from a suitable perhydrate (such as sodium percarbonate).The present application suggests the use of further substrates to helpbring about the reaction to produce hydrogen peroxide by means ofsupplemented enzymatic catalysis. Hydrogen peroxide is toxic to bothbacterial and mammalian cells, while hypothiocyanous acid reacts withfree sulphydryl groups in bacterial proteins, inactivating severalmetabolic enzymes.

The consensus in the literature teaches that the LP system has mainly abacteriostatic effect on catalase positive gram positive bacteria andalso that there is a pH dependent bactericidal effect on some gramnegative bacteria (Wofson and Sumner, 1993, “Antibacterial activity ofthe lactoperoxidase system: a review” Journal of Food Protection 1993,56(10):887-892; Kussendrager and van Hooijdonk, 2000 “Lactoperoxidase:physico-chemical properties, occurrence, mechanism of action andapplications”, British Journal of Nutrition, 84, Suppl. 1, S19-S25;Seifu et al., 2005 “Significance of the lactoperoxidase system in thedairy industry and its potential applications: a review”, Trends in FoodScience & Technology 16, 137-154). The precise mechanisms underpinningthe antimicrobial properties of the LP system remain unresolved. Theexperimental protocols reported in the literature vary widely and manyauthors report bacterial inhibition, but regrowth after a particularperiod of time, and thus a biostatic rather than a biocidal effect (e.g.Ishido et al. “Continuous supply of OSCN-ions by lactoperoxidase systemdeveloped from lactose as the primary substrate and its anti-bacterialactivities”, Milchwissenschaft, 66 1, 2011) Documents have also claimedbactericidal activity using the LP system, but have reported significantnumbers of culturable cells remaining after testing, even at elevated LPconcentrations (e.g. Garcia-Garibay et al., 1995, Antimicrobial effectof the lactoperoxidase system in milk activated by immobilised enzymes”Food Biotechnology, (3), 157-166). As discussed elsewhere in thisapplication, hydrogen peroxide, either provided in the medium orproduced during the LP reaction, is toxic to bacterial cells and, unlessspecific controls are in place, it may contribute to reportedantimicrobial activity. The prior art has also disclosed a range ofreportedly synergistic compounds, which are claimed to either enhance,or indeed enable, the effectiveness of the LP system. In addition, USpatent application number 2011/0008361 A1 suggests that theantimicrobial effect of the described extracted cationic fractions,including LP, are a combination of immune stimulation, which helps toclear infection and direct antimicrobial activity. The lack ofsystematic information on the precise factors influencing theantimicrobial effects of the LP system is a barrier to commercialapplications—as is the potential for hydrogen peroxide toxicity insensitive settings, such as the mammalian lung.

While the LP system has been widely described, it is thought by thoseskilled in the art that this is an ineffective system to be utilised ofthe treatment of bacterial infection (Rainard & Riollet 2006, “Innateimmunity of the bovine mammary gland”, Veterinary Research 37, 369-400;Sakai et al., 2008, “Generation of hydrogen peroxide by a low molecularweight compound in whey of Holstein dairy cows”, Journal of dairyResearch, 75, 257-261; Sakai et al., 2008, “Production of hydrogenperoxide by a small molecular mass compound in milk from Holstein cowswith high and low milk somatic cell count”, Journal of Dairy Research,75, 335-339). A lack of robust and effective bactericidal capacity ofthe LP and similar systems is a significant barrier to suchapplications. Current commercial applications based on the LP systeminclude mouthwash, toothpaste, food preservation and disinfectants,which are mainly based on inhibition of microbial growth, rather thanthe killing of cells and the total elimination of bacterial populationsfrom various settings. For example, the patents filed regarding thelactoperoxidase system (Number U.S. Pat. Nos. 4,726,945 and 5,607,681)are used for topical treatments and are designed mainly as a way toreduce the growth of bacteria present (either in the solutions, forexample as an ionic emulsion, or acne or athlete's foot treatments, orindeed as a prophylactic in feedstuffs for animals).

The broad spectrum and numerous potential targets of the antimicrobialspecies of the invention are unlikely to induce the proliferation ofresistance genes. Drug reactions would not be a problem either, beingthat components of the therapeutic composition may be produced naturallyin the mammalian body (for example, via intermediates such asthiocyanate, lactoperoxidase, glucose). Reaction to drugs is a currentproblem, with 20% of patients reacting to B-lactam drugs (“Rapidde-sensitization for non-immediate reactions in patients with cysticfibrosis”, Whitaker et al., 2011, Journal of Cystic Fibrosis,10(4):282-5).

Ishido et al. (Milchwissenschaft, Vol. 66, No. 1, 2011) describe alactoperoxidase system comprising lactoperoxidase, glucose oxidase andβ-galactosidase, lactose and potassium thiocyanate as an agent, which issaid to be antibacterial. In all cases lactose was present in thecomposition. The document, however, describes bacterial growthsuppression only and no analysis was conducted for more than 48 hours.The document also states that there were no inhibitory effects againstthe gram-negative bacteria E. coli and Klebsiella pneumoniae and thegram-positive species, S. xylosus, E. faecalis, E. faecium and E.raffinosus. The results in the document are labelled as being ‘growthsuppression’ times, the time tested being no more than 12 hours. Overallthis paper thus describes a bacteriostatic effect and not a bactericidaleffect.

Garcia-Garibay et al. (Food Biotechnology, 9 (3), 157-166 (1995)describes the use of β-galactosidase and glucose oxidase to producehydrogen peroxide in raw milk, to reduce undesirable micro organisms inthe milk. Again testing was conducted for 24 hours only, and thus thedata presented showing a reduction in microorganism numbers indicatesonly a bacteriostatic effect.

Sandholm et al. (J. Vet. Med. B 35, 346-352 (1988) describes glucoseoxidase for use as an antibacterial agent in teats or intramammaryantiseptics.

U.S. Pat. No. 5,607,681 describes antimicrobial compositions comprisingiodide and thiocyanate ions, an oxidoreductase enzyme and itscorresponding oxidisable substrate, together with a lactoperoxidase.This document only describes activity against bacteria for up to 72hours and describes no therapeutic application of the system.

The present invention represents a paradigm shift from the prior art. Itdescribes a broad-spectrum, truly microbiocidal, therapy for treatmentof infections.

-   -   The present inventors have shown that the reactive oxygen        species (ROS) produced by the LP system, or by other means are,        in fact, bactericidal to a wide range of pathogenic        gram-positive and gram-negative bacteria, in a variety of media        and across a wide range of pH and temperature ranges, based on        concentration dependent dose response (see detailed description        of the invention). We have also shown that the ROS can        completely kill bacteria and fungi growing in biofilms on        various surfaces.    -   In a detailed series of controlled trials, it has been shown        that this bactericidal activity can be exclusively due to the        action of the ROS and that the ROS are effective in this regard        with no residual hydrogen peroxide present and in the absence of        any other synergistic agents such as lactoferrin, quarternary        ammonium compounds, fatty acids, etc (see detailed description        of the invention).    -   The concentrations of ROS required to completely kill particular        populations of bacteria in particular settings can be calculated        and used to generate precise dose response/minimum inhibitory        concentration information, in a manner similar to that used for        antibiotics and other antimicrobial therapeutic agents (see        detailed description of the invention).    -   Administration of the ROS species at these dose levels can be        used to completely kill a wide variety of gram negative and gram        positive bacterial pathogens—including those isolated from the        bovine udder and mammalian lung, those growing as biofilms        attached to various surfaces; those isolated from patients; and        those that are resistant to a wide range of antibiotics (see        detailed description of the invention).    -   It has also been shown that the required bactericidal and        therapeutic concentrations of the ROS species can be generated        at the site of treatment, for example using the LP system in the        bovine udder, or that the ROS species can be prepared externally        to the site of infection and delivered in the absence of any        other active ingredient to allow successful treatment and        elimination of bacterial infections.

The present technology thus provides a unique way to limit microbialinfection and biofilm growth on the surface of, for example, medicaldevices.

OBJECT OF THE INVENTION

A first object of the invention is to provide an improved compositionfor the treatment of microbial infections. A further object is toprovide a composition which is capable of killing, as opposed to slowingthe growth of, bacteria, viruses, fungi and yeasts. A still furtherobject is to provide a composition that can kill antiobiotic resistantorganisms.

Mastitis

A further objective is to provide a composition for the treatment ofboth clinical and sub-clinical mastitis, without the use of antibiotics.A further object of the invention is to provide a treatment formastitis, which does not require that the milk be discarded duringtreatment.

A further object of the invention is to provide a clinical solution tomastitis, which would be easy to administer. Such is the reaction in theprior art discussed above, that if all the components are mixedtogether, the reaction would occur instantly, thus leaving a shortshelf-life. In the prior art it is necessary to have two aliquots thatare mixed prior to administering the product, thus starting thereaction. Another drawback is the fact that the glucose concentration isalways a limiting factor. To have enough glucose in a clinicalsituation, a concentrated source needed to be added. This leads tosolubility problems. The addition of extraneous glucose may also lead toissues in the downstream processing of the milk and may also lead toconsumer problems regarding the taste and sweetness of the milk forconsumption.

In an attempt to circumvent the need for glucose administration, thecharacteristics of milk itself were examined by the present inventors.Milk is a source of lactose. Beta-galactosidase is an enzyme thatcleaves and converts the disaccharide sugar into its constituents,glucose and galactose. The use of the enzyme would therefore rely on akey constituent of milk itself to exploit the ability of the LP systemto generate the reactive oxygen species at the site of infection. Usingthis enzyme makes it easier to administer the cocktail as the systemwill not react until glucose is present (and glucose would not beproduced until Beta-galactosidase was in contact with the milk). This ismuch more useful than having to administer glucose or indeed a suitablemonosaccharide or disaccharide sugar prior to usage, negating the use ofa concentrated and problematic sugar solution.

Lung Infection

Another object of the invention is the treatment of a number of otherbacterial infections. Drug delivery and resistance to antibiotics is amajor problem in the treatment of both cystic fibrosis (CF) andtuberculosis (TB). Antibiotic resistance often occurs as a result ofonly sub-inhibitory concentrations of the drug reaching the target site,i.e. lungs. Chronic infections will often result from this situation,seriously impairing the health of the patient. The ideal delivery of thecomponents of the lung would be as a nebulised spray, directly into thelungs. This would have the advantage that the components would interactdirectly with the organism at the correct site, and at the correctconcentration. If it were administered orally or through the bloodstream, greater concentrations are required to give the same effect.When using antibiotics, this will lead to large concentrations of thedrug being used, increasing the chances of resistance or a reaction tothe drug. Because the components of the present system are presentnaturally in the mammalian system (or used in feedstuffs regularaly),reaction to them is highly unlikely, an advantage to treatment withantibiotics where drug reactions are common. In patients with CF, thenatural level of thiocyanate is reduced in the lung due to the failureof regulation of water in the membrane, reducing the effectiveness ofthe naturally found lactoperoxidase system in the organ. Extraneousaddition of the reactive oxygen species that could act propylacticallyon people diagnosed with CF or be used to treat patients that havealready developed lung infections. Such a nebulised spray has thepotential to be used in hospitals in minimising infection duringoperations where body cavities are open to the environment and at risk,especially those caused by antibiotic resistant strains of bacteria.

Burns/Skin/Mouth

Other infections that are suitable targets for the technology are thoseincurred as a result of burns or an open wound. The advantage of usingthe described system over present antibiotic treatment regimens would bethe similar to those described above in treating CF or TB as regardsdrug reaction, safety, efficacy, and resistance. The inventors haveshown that the reactive oxygen species are effective in the treatment ofbiofilm based cells (those attached to a solid stratum). This would bethe bacterial phenotype most noted in the lungs of TB/CF patients, andin open wounds or burns. This phenotype confers a generalised toleranceto the bacteria against a wide range of antibiotics (“Antibioticresistance of bacteria in biofilms”, Stewart & Costerton, 2001, TheLancet, 358, 135-138).

A form of the system could also serve as a general antibacterialsolution, having numerous purposes. For example, a mouthwash containingthe antibacterial system could help prevent the formation of biofilmswithin the mouth. Likewise, an antibacterial nasal rinse could be usedto help alleviate sinus problems, such as sinusitis or allergicrhinitis. Currently, steroids and saline nasal washes are used. Anantibacterial saline wash would, however, also help to combat bacterialcolonisation within the nasal cavity.

Medical Devices

The use of reactive oxygen species, such as those generated by ahaloperoxidase based system, would also hold a number of advantages inthe treatment and prevention of bacterial infections on an implantedmedical device. Infections (in the form of a recalcitrant biofilm) areextremely common in various devices such as catheters. A deviceimpregnated or coated with the various enzymes would be able to reactwith substrates naturally present in the blood.

General Antibacterial Wash

Another object of the invention is to provide a composition for use as ageneralised, safe, antibacterial wash for a multi-purpose product. Theproposed system could be effective at washing/removal of bacteria fromsurfaces, pipes, beer lines, cooking equipment etc.

Antifungal Agent

Infections can occur as a result of yeast or fungal growth as well asbacteria. As such, a therapeutic regime capable of exertingantimicrobial activity (as opposed to antibacterial activity, as is thecase with antibiotics) would be of great benefit. To this end, theantimicrobial activity of the reactive oxygen species (hypoiodate) wastested against two fungal strains of note (Example 18 below). TheCandida strains of fungus are typically described as opportunisticpathogens. They can cause a variety of skin conditions, such asvulvovaginitis and urinary tract infections. They are prevalent in HIVpatients, and other immunocompromised patients. Saccharomyces cerevesiae(commonly known as yeast, or bakers' yeast) is an important organism infood production. It also poses great problems for the drinks industry,and is a typical organism found on beer lines, and is the cause ofbeverage spoilage. As such, the antimicrobial reactive oxygen species ofthe invention, would be a good therapeutic candidate to treat or controlfungal infections, or to clear surfaces, eradicating fungalcontamination.

The invention also finds use as an anti-viral agent.

SUMMARY OF THE INVENTION

According to the present invention there is provided a microbiocidalcomposition comprising a reactive oxygen species or components capableof producing a reactive species, the composition being capable ofdelivering the reactive oxygen species to a level of at least 0.4millimoles per litre over a 24 hour period. The composition may becapable of delivering the reactive oxygen species to a level of at least0.5 millimoles per litre, or at least 0.6 millimoles per litre or atleast 0.7 millimoles per litre or at least 0.8 millimoles per litre orat least 0.9 millimoles per litre or at least 1.0 millimoles per litreor at least 2.0 millimoles per litre over a 24 hour period.

The reactive oxygen species may be produced by the reaction of aperoxidase, a substrate for the peroxidase and hydrogen peroxide.

By microbicidal we mean a composition which is capable of killingmicrobes such as bacteria, viruses, fungi and yeasts, as opposed tosimply retarding their growth. The microbicidal composition is capableof killing at least 90%, preferably at least 95%, more preferably atleast 99%, more preferably at least 99.99%, of the microbes present inan environment to which it is applied.

The compostion is also capable of killing antiobiotic resistantorganisms.

The reactive oxygen species of the composition may comprisehypothiocyanate (also known as hypothiocyanite, SCNO⁻).

The reactive oxygen species of the composition may also comprisehypoiodate (IO⁻ also known as hypoiodite).

The reactive oxygen species of the composition may also comprisehypochlorite (ClO⁻).

In a further aspect the invention provides a microbicidal compositionadditionally comprising a peroxidase enzyme and an oxidoreductase. Thiscomposition is particularly suitable for the treatment of infections incystic fibrosis or tuberculosis patients, burns victims or patients withinserted medical devices.

In a further aspect the invention provides a microbicidal compositionadditionally comprising a peroxidase enzyme, an oxidoreductase, and aglycoside hydrolase. This composition is particularly suitable for thetreatment of mastitis infections.

The composition may also comprise a substrate for the peroxidase.

The peroxidase may be a haloperoxidase. The haloperoxidase enzyme may bea lactoperoxidase, a chloroperoxidase, a bromoperoxidase or aniodoperoxidase. Suitable chloroperoxidases include myeloperoxidase andeosinophil peroxidase. Suitable bromoperoxidases include ovoperoxidase,vanadium bromoperoxidase and Murex snail bromoperoxidase. Suitableiodoperoxidases include horseradish peroxidaseand thyroid peroxidase.

If a lactoperoxidase enzyme is used, the composition may furthercomprise potassium or sodium iodide or potassium or sodium thiocyanateas the substrate. Chloroperoxidase reacts with chloride ions, readilyavailable in milk, blood or the like, so the addition of chloride ionsmay not always be necessary. If bromooxidase or iodooxidase are used,the substrate may be a source of bromide or iodide ions, respectively.

The haloperoxidase reacts with available hydrogen peroxide, and asuitable substrate (iodide/bromide/chloride or thiocyanate) to producean antibacterial reactive species.

The glycoside hydrolase may be Beta-galactosidase, which converts freelyavailable lactose in the milk into glucose and galactose.

The oxidoreductase may be glucose oxidase, which reacts with theresulting glucose to produce hydrogen peroxide. Similarly, galactoseoxidase could also be used, reacting with galactose to produce hydrogenperoxide.

Preferably, the composition may further comprise a disaccharide sugar.The disaccharide could be subsequently hydrolysed by its correspondingglycoside hydrolase, for example sucrose and sucrase, allowing therelease of monosaccharide sugars, which in turn could act as a source ofadditional hydrogen peroxide by the use of a corresponding oxireductaseenzyme.

Additionally, oligosaccharides or polysaccharides containing more thantwo sugar molecules, and that are cleaved to produce a source ofhydrogen peroxide may be added.

In some embodiments, the composition comprises two enzymes to derive asource of hydrogen peroxide; a glycoside hydrolase to break downdisaccharide sugars into constituent monosaccharides, and a furtheroxireductase enzyme that reacts with the monosaccharide sugars torelease hydrogen peroxide.

The composition may comprise additional sources of hydrogen peroxide.One additional source of hydrogen peroxide is the exogenous addition ofa solution of hydrogen peroxide or its release by a suitable perhydrate,such as sodium percarbonate. Alternatively, or in addition, a number ofenzymes can be used to produce hydrogen peroxide. Xanthineoxidoreductase/oxidase reacts with either hypoxanthine or xanthine (bothpresent in milk) to produce hydrogen peroxide. Therefore, xanthineoxidoreductase/oxidase and/or xanthine could be added to the compositionproducing hydrogen peroxide. Similarly, sugar alcohols can be reactedwith their appropriate oxidase enzymes to produce a source of hydrogenperoxide. For example, glycerol oxidase reacts with glycerol to producea source of hydrogen peroxide and, therefore, glycerol oxidase/glycerolcould be added to the composition. Another example would be mannitolreacting with mannitol oxidase. Further to this, citric acid is known torelease hydrogen peroxide, and therefore could also be added to thecomposition. Similarly, L-amino acid oxidase is an enzyme that reactswith free amino acids (also present in milk) to produce hydrogenperoxide. Likewise, its addition (with or without L-amino acidsupplementation) could provide a source of hydrogen peroxide.

The aspect of the invention, which provides a bactericidal compositionadditionally comprising a peroxidase enzyme, an oxidoreductase, and aglycoside hydrolase (specifically Beta-galactosidase), has proven tohave extremely effective antibacterial qualities in milk. In particular,the system is effective at completely killing both Gram-positive andGram-negative organisms at high levels of bioburden (i.e. eliminating10⁸⁻¹⁰ cells/ml). Based on the prior art, this is a surprising findingas the WHO have stated that the lactoperoxidase system ‘exerts primarilya bacteriostatic effect in raw milk’ and was suitable only for limitingbacterial growth on a short term basis by such a means during thetransport of raw milk, in the absence of refrigeration (Report of anFAO/WHO Technical Meeting FAO Headquarters, Rome, Italy, 28 Nov.-2 Dec.2005). Other researchers concluded that any bacteriocidal activity inthe system was due to the hydrogen peroxide component and that thelactoperoxidase system was bacteriostatic (Thomas et al., 1994,“Antibacterial activity of hydrogen peroxide and thelactoperoxidase-hydrogen peroxide-thiocyanate system against oralstreptococci”, Infection and Immunity, Vol. 62, No. 2, p 529-535).

The Beta-galactosidase reacts with lactose present in milk, to produceglucose and galactose. The resulting glucose reacts with the glucoseoxidase to produce hydrogen peroxide. The hydrogen peroxide reacts withpotassium iodide/thiocyanate to produce an antibacterial effect. Theantibacterial effect is aided by lactoperoxidase which catalyses theperoxidation of iodides and other suitable substrates.

Relying on the inherent lactose in milk negates the use of a highlyconcentrated glucose solution, which was a limiting factor in an invitro situation, making it possible to use the product in the field.

In another aspect of the invention, there is provided a composition forthe treatment of infections or contamination, comprising Xanthineoxidoreductase/oxidase. The composition may further comprise eitherhypoxanthine or xanthine or both. The composition may also comprise anoxidoreductase or a glycoside hydrolase or disaccharides.

In yet another aspect of the invention there is provided a compositionfor the treatment of infections or contamination comprising L-amino acidoxidase. The composition may further comprise free amino acids. Thecomposition may also comprise an oxidoreductase or a glycoside hydrolaseor disaccharides.

One potential of the composition of the invention lies in the treatmentof bovine (or other mammal) mastitis. It could be used to treat bothclinical and sub-clinical mastitis and would provide the advantage ofnot requiring the removal of resulting treated milk from the bulk pool.The enzymes present in the proposed solutions are safe and the potentialsubstrates, including potassium iodide or thiocyanate are safe at theconcentrations used. The composition is unlikely to induce resistance toantibiotics, which is an additional advantage. In addition, the reportof an FAO/WHO technical meeting in Rome, Italy on 22 Nov.-2 Dec. 2005indicated that a lactoperoxidase system does not introduce substancesinto milk that are not normal metabolites.

The use of enzymes to produce the biocidal reactive oxygen species ofthe invention, provides the means to continuously produce thecomposition over periods of time, which is advantageous when compared toantibiotic treatments, where new molecules must be externally added toreplenish the antimicrobial activity.

One product suitable for the treatment of mastitis is an intramammaryinfusion delivery device (optionally presented as a dual barrelledsyringe) loaded with 7-10 ml solution (increasing its viscosity bygelatine supplementation) containing 2 mg glucose oxidase (˜200units/mg), 0.5 ml Beta-galactosidase (≥2,600 units/ml), 4 mglactoperoxidase (≥80 units/mg), and 100-150 mg potassium iodide, to beused to generate the bactericidal reactive oxygen species to treatmastitis.

Other preparations could hold the enzymes as a lyophilized powder to bemixed with a solution of substrates prior to use, thus aiding shelf-lifeof the reaction where refrigeration is not a possibility. Other similarproducts may be produced based on the weight of animal (sheep, etc.),milk production, and the bioburden level in the infected udder. Furtherto this, various combinations of components could also be prepared,notably using bromide/thiocyanate instead of iodide, using a differentoxidoreductase enzyme, or the supplementation of different possiblesources of hydrogen peroxide, or to use a xanthine oxidoreductase (acomplex enzyme which comprises xanthine oxidase) or an L-amino acidoxidase (a member of the oxidoreductase enzyme family) approach.

An alternative product involves substitution of lactoperoxidase with anyother enzyme that reacts with hydrogen peroxide and suitable substrateto produce the antimicrobial species. Of these, chloroperoxidase issuitable with a loading of 7-10 ml solution (increasing its viscosity bygelatine supplementation) containing 2 mg glucose oxidase (˜200units/mg), 0.5 ml Beta-galactosidase (≥2,600 units/ml), and 50 μlchloroperoxidase (≥11,100 units/ml).

A lactoperoxidase system has been described before (Number U.S. Pat.Nos. 4,726,948 and 5,607,681) in a number of formulations. The WorldHealth Organisation (Report of an FAO/WHO Technical Meeting FAOHeadquarters, Rome, Italy, 28 Nov.-2 Dec. 2005) has recommended its useas a method of increasing the longevity of milk in the absence ofrefrigeration in 3^(rd) world countries. The WHO recommended a systemusing sodium percarbonate as the source of hydrogen peroxide. The aspectof the invention presented here differs from the known LP system in thatit uses an alternative source of hydrogen peroxide, provided by thesequential cleavage of lactose present in the milk. Further to this, analternative is to use chloroperoxidase enzyme in place oflactoperoxidase, wherein, it reacts with salt already present in milk,negating the need for halide supplementation to the udder. Either formof this aspect of the invention (lactoperoxidase/chloroperoxidase)offers the advantage over existing preparations in that they would notstart reacting until administered to the udder. Each would have alengthy shelf-life at 4° C. The WHO discussed a number of problems intheir described incarnation of the LP system that would not occur in theaspect of the invention described herein. The system proposed by the WHOdelivered thiocyanate and percarbonate powders in sachet form to milk.Powdered thiocyanate is hygroscopic and may degrade overtime, and sodiumpercarbonate as a source of hydrogen peroxide may lead to oxygenproduction, which could cause rupture and breakage of the sachet.

The invention described here improves on the lactoperoxidase systemdescribed by Kussendrager and van Hooijdonk, 2000 (Lactoperoxisdase:physico-chemical properties, occurrence, mechanism of action andapplications. British Journal of Nutrition, 81, 519-525). When treatingmastitis, it uses a key ingredient of milk itself, lactose, to drive thereaction to produce the specific required concentrations of thebactericidal reactive oxygen species to eliminate the infection. Thesupplementation of two enzymes (Beta-galactosidase and glucose oxidase)as opposed to sodium percarbonate, allows a slow, prolonged, release ofhydrogen peroxide necessary to allow continuous production of thebactericidal agents at a controlled level. Because of the availabilityof lactose in the proposed environment, hydrogen peroxide would not be alimiting factor in the reaction. A relatively small amount ofBeta-galactosidase has proved just as effective as larger volumes ofglucose supplementation.

The invention improves on the lactoperoxidase system found naturally inmammals as it regulates the levels of hydrogen peroxide present (andindeed any other components). During a typical infection, the level ofhydrogen peroxide is quenched by bacterial catalase activity, thusreducing the effectiveness of the system. The low availability ofhydrogen peroxide has lead a number of researchers to believe theantimicrobial nature of the lactoperoxidase system was ‘questionable’(Rainard & Riollet 2006, “Innate immunity of the bovine mammary gland”,Veterinary Research 37, 369-400; Sakai et al., 2008, “Generation ofhydrogen peroxide by a low molecular weight compound in whey of Holsteindairy cows”, Journal of dairy Research, Journal of Dairy Research, 75,257-261; Sakai et al., 2008, “Production of hydrogen peroxide by a smallmolecular mass compound in milk from Holstein cows with high and lowmilk somatic cell count”, Journal of Dairy Research, 75, 335-339).

The embodiment of the invention which uses xanthineoxidoreductase/oxidase or L-amino acid oxidase as sources of hydrogenperoxide would improve the technology as neither require sugarsupplementation, use constituents of the milk itself, and do not alterthe taste of the milk as regards its sweetness.

The patents filed regarding the lactoperoxidase system (U.S. Pat. Nos.4,726,945 and 5,607,681) are used for topical treatments and aredesigned mainly as a way to reduce the growth of bacteria present(either in the solutions, for example as an ionic emulsion, or acne orathlete's foot treatments, or indeed as a prophylactic in feedstuffs foranimals). The present invention provides a novel means to treat“full-blown” bacterial infection as an alternative to antibioticregimens. High levels of bactericidal activity have been shown using theinvention, in its various aspects, both at an in vitro and in vivolevel.

Variations on the technology, namely the type of enzymes and substratesutilised to produce the bactericidal reactive oxygen species, would beused to treat other types of bacterial infection.

An antimicrobial nasal rinse product in accordance with the inventioncontains the required components of the system. These include a suitableperoxidase enzyme, such as lactoperoxidase or chloroperoxidase, theappropriate substrate (such as iodide, thiocyanate, or chloride ion).Similarly, a source of hydrogen peroxide would be supplied, optionallyby a mono-saccharide sugar and its cleaving enzyme, for example glucoseand glucose oxidase, or a hydrogen peroxide releasing molecule, such aspercarbonate or citric acid. These components and substrate(s) may besupplemented as a dry salt powder/lyophilised enzyme in a sachet thatwould be re-hydrated before use. The use of saline solution (or theaddition of extra sodium chloride to the sachet) would regulate therequired salinity. Likewise, the use of sodium bicarbonate wouldregulate the acidity. A dry-powdered form of the system would allow theproduct to maintain shelf-life, reduce the volume of the product so thatonly water would be needed to ‘activate’ the system.

A composition for the treatment of CF/TB may be a solution delivered tothe lung by means of a nebulised spray, which would deliver the requiredcomponents of the invention (either lactoperoxidase or chloroperoxidase,required substrate (thiocyanate/chloride), and a source of hydrogenperoxide (glucose and glucose oxidase, or other possible enzymaticmethods, such as xanthine oxidoreductase/oxidase andhypoxanthine/xanthine or L-amino acid oxidase and L-amino acids, or bythe addition of a perhydrate such as sodium percarbonate, or even by thedirect addition of a hydrogen peroxide solution) to the lung. Storage ofthe solution may be achieved by separating certain components to ensurethe reaction would only occur on delivery to the lung. As such, onesolution may contain the haloperoxidase enzyme and glucose and would bemixed with another solution containing the substrate and glucoseoxidase. Mixing of these would start the reaction, producing the highlyantibacterial reactive species that could be delivered to the lung bynebuliser in a given volume. Concentrations of each of the componentsmay be tailored to the human lung, but would be of the same possibleorder of magnitude as those described for the mastitis treatment above.Likewise, the same overall delivery mechanism may be used for a generalantibacterial throat spray, which would not require the need fornebulisation.

The ability to efficiently eradicate biofilm cells by the presentinvention (see detailed description of the invention Example 3) confersa significant improvement over presently used antimicrobial treatmentsthat are known to be inefficient in the treatment of cystic fibrosisinfection of the lung as Pseudomonad strains exhibit greater toleranceto antibiotics (“Differences in biofilm formation and antimicrobialresistance of Pseudomonas aeruginosa isolated from airways ofmechanically ventilated patients and cystic fibrosis patients”,Fricks-Lima et al., 2011, International Journal of Antimicrobial Agents,37(4), 309-315).

One embodiment of the invention also provides a new method of treatingburns patients. A poultice impregnated with the enzymes capable ofproducing the antimicrobial species (notably a haloperoxidase; such aslactoperoxidase/chloroperoxidase; and an oxidoreductase, such as glucoseoxidase) may be dipped into a gel based solution containing ingredientsnecessary for the production of antibacterial compounds; the potentialsubstrate, notably iodide/thiocyanate/chloride; and a monosaccharidesugar that will react with the oxidoreductase enzyme, notably glucose.This poultice could be placed over the wound where required, allowing asafe release of highly antibacterial compounds, maintaining a safe,aerobic environment needed for tissue repair. Concentrations of thecomponents would again be similar to those described above for thetreatment of mastitis. Alternative methods of producing hydrogenperoxide could be used by the varying of the oxidoreductase and sugar,by other enzymatic reactions (L-amino acid oxidase and L-amino acids,xanthine oxidoreductase/oxidase and xanthine/hypoxanthine), by theaddition of a perhydrate such as sodium percarbonate, or the directaddition of a solution of hydrogen peroxide.

The embodiment of the invention utilising the haloperoxidase basedsystem may also be employed to improve the physical properties ofmedical devices, to limit the chance of causing infection duringimplantation into a patient. The method is safe, limiting drug reactionor resistance, as is often the case in using antibiotics. Theimpregnation of required enzymes, lactoerpoxidase or chloroperoxidase,and an oxidoreductase, glucose oxidase and/or additional substrates,onto the surface of the device, is a viable method of delivery.Typically, the substrates required to drive the reaction, notablyglucose and thiocyanate/chloride are present in blood, saliva, milk, andurine, thus a reaction would occur on the slow release of the enzymesand/or additional substrates. The slow release of the enzymes may beundertaken by their anchoring/coating on the surface by means ofelectrical charge, in the form of an impregnated biodegradable polymer.As the polymer degrades, fresh enzyme molecules are free to react withsubstrates passing over the device. The steady release of these enzymesover the course of the initial number days, post implantation, wouldmaintain sterility of the device in the important window of opportunitywhere infections/biofilms would normally take hold.

A generalised antibacterial solution may be produced by the mixing oftwo solutions (or possibly their dried-powder forms in water). Forexample, an antibacterial solution may be prepared by the mixing ofcrude sources of peroxidase enzyme (chloroperoxidase or lactoperoxidase,for example), the appropriate substrate (optionally in the form of asalt for example sodium iodide/thiocyanate/or chloride) and a source ofhydrogen peroxide (cheap sources would include citric acid,percarbonate, hydrogen peroxide itself, or sugars with the appropriateenzyme to react with it—for example, glucose and glucose oxidase). Thisaspect on the invention would be activated by the mixing of thecomponents in solution. The solution may be delivered to the surfaceneeding to be cleaned. Such examples would include beer lines, ceramicsurfaces, metal surfaces, etc. and could be used in hospitals,factories, kitchens and the like. The solution would be relativelynon-toxic, and would not produce the odour associated with many cleaningproducts such as bleach. Further to this, a protein such as lactoferrincould be supplemented to the system to increase potency. Lactoferrin isknown to help break down bacterial biofilm, and thus may serve toaccentuate the antimicrobial nature of the product.

Likewise, the proposed compositions could be pre-prepared or activatedbefore administration to an infection site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the susceptibility of E. coli to inhibition by H₂O₂.Growth, measured by optical density at 595 nm, was inhibited atdilutions of 1/8 and 1/16, but not 1/32.

FIG. 2 depicts the susceptibility of E. coli to H₂O₂ in the presence ofKI/LP. Growth, measured by optical density at 595 nm, was inhibited atdilutions of 1/8 and 1/16, but not 1/32.

FIG. 3 depicts the susceptibility of E. coli to peroxide released by theaddition of percarbonate. Growth, measured by optical density at 595 nm,was inhibited at dilutions of 1/8 and 1/16, but not at a dilution of1/32.

FIG. 4 depicts the susceptibility of E. coli to peroxide released by theaddition of percarbonate in the presence of KI and LP. Growth, measuredby optical density at 595 nm, was inhibited at dilutions of 1/8 and1/16, but not at a dilution of 1/32.

FIG. 5 depicts the susceptibility of E. coli to peroxide produced fromGlucose by enzymatic activity via glucose oxidase.

FIG. 6 depicts the susceptibility of E. coli to peroxide releasingenzymatic glucose and glucose oxidase in the presence of supplemented KIand LP.

FIG. 7 depicts the proposed schematic model of hydrogen peroxide levelsover time.

FIG. 8 depicts the standard curve of thiocyanate levels against opticaldensity.

DETAILED DESCRIPTION OF THE INVENTION Example 1

An antimicrobial composition was produced by the addition of 150 mgpotassium iodide, 4 mg lactoperoxidase (>80 units/mg), and 2 mg glucoseoxidase (˜200 units/mg) to 7 ml sterile water. This embodiment isreferred to as ‘KI-Dose-150’. Similarly, a composition was preparedusing 150 mg potassium thiocyanate, and is referred to as‘Thio-Dose-150’. The antimicrobial properties of these compositions weretested using a doubling dilution 96-well plate growth assay-basedmethod. An aliquot of the composition was added to 150 μl growth medium(with 1-2% glucose present) containing 10⁷⁻⁸ bacterial cells, andbrought to a final volume of 300 The composition concentration wasdoubly diluted by the removal of 150 μl of the mixture and transfer tothe next well containing 150 μl of identical growth medium, lacking theROS producing components. The optical density of the medium was measuredfor 24 hours at 595 nm. The concentration required to completely inhibitthe bacteria was the lowest concentration of the composition employedthat resulted in no visible signs of growth in the medium after the 24hours. Controls included wells to which none of the composition wassupplemented, or wherein, one of the components was removed from thecomposition. This method was used to determine the antimicrobial potencyof the compositions against a variety of micro-organisms, notablyEscherichia coli, Staphylococcus aureus, Psuedomonas aeruginosa,Burkholderia cepaciae, Streptococcus dysglactiae, Streptococcus uberis,a non-haemolytic coliform. These organisms are the often described ascausative agents of numerous infections, notably bovine mastitis, cysticfibrosis lung infections, skin infections, burns infections, etc and, assuch, represent the variety of organisms that the composition will beused to treat. The relative susceptibility of the organisms to thecompositions is described in Table 1 (Ratio indicates the lowestdilution of the composition at which no growth was still recorded, forexample, 1:800 had the composition diluted to the equivalent of 1 μlcomposition for every 800 μl of growth medium) and are the lowestconcentrations of the composition that inhibited growth. The estimatedlevel of the antimicrobial reactive oxygen species (ROS) produced overthe course of 24 hours is also provided (see also Example 19 below).

TABLE 1 Susceptibility of bacterial strains to ‘Thio-Dose-150’ and‘KI-Dose-150’. Thiocyanate Iodide Strain Dilution ROS MIC Dilution ROSMIC E. coli ATCC 25922 1:400 0.4-0.8 1:800   0.25-0.5 Strep. dys 143  1:48,000 0.003-0.007 1:48,000 0.005-0.01 Strep. dys 160   1:48,0000.007-0.01  1:48,000 0.005-0.01 Strep. uberis   1:24,000 0.007-0.01 1:24,000 0.005-0.01 Staph. aureus 15676 1:800 0.3-0.6 1:800   0.25-0.5Burk. cepacia 1:800 0.3-0.6 1:1,600  0.075-0.15 P. aeruginosa PA01 1:8000.3-0.6 1:1,600  0.075-0.15 Non-haemolytic coliform 1:400 0.4-0.81:800   0.25-0.5 The MIC indicates the level of reactive oxygen speciesbelow which bacteriocidal effects were not noted (millimoles per litreproduced over 24 hours).

Example 2

The lactoperoxidase system has been discussed in terms of itsbacteriostatic or inhibitory qualities. The protocol described inExample 1 was performed in larger scale volumes (an initial 500 μlaliquot of KI-Dose-150 or Thio-Dose-150 was added to 10 ml growth mediumcontaining the test organism and doubly diluted). After 48 hours ofincubation, the bacteriocidal qualities of the system were investigatedby the sub-culture of the inoculated broths to agar plates. Thecomposition was deemed to be bacteriocidal for a bacterial strain at aparticular concentration if no more than 0.0001% of cells wererecoverable after 24 hours following sub-culture to the fresh agar plate(ie 72 hours after exposure to the antimicrobial composition). Thecompositions were bacteriocidal to each of the tested strains atconcentrations that would be achievable for an infection treatment(lowest dilutions at which inhibition was noted are presented in Table 1above.

Example 3

The choice of hydrogen peroxide source can be shown to affect the the LPsystem and also the ability to produce an antimicrobial compositionbased on ROS at bactericidal concentrations. Aliquots (20 ml) ofnutrient broth were inoculated with 10⁸ cells of E. coli ATCC 25922, and150 μl of the inoculated broth were added to wells in a 96 well growthplate. This was repeated with the inoculated broth being furthersupplemented with LP (37.5 μl of a 4 mg/ml solution) and KI (75 μl of a40 mg/ml solution) to the medium.

Sources of hydrogen peroxide were added (150 μl) to well 1 and doublydiluted to well 11, but not to well 12, which acted as a control. Thesources of hydrogen peroxide were as follows:

-   -   5 ml water containing 40 μl H₂O₂ (30% w/v)    -   5 ml water containing 50 mg sodium percarbonate    -   5 ml glucose (20%)+47 μl glucose oxidase (2.5 mg/ml, 200        Units/mg)

FIG. 1 illustrates that 1:8 and 1:16 dilutions of the H₂O₂ solution wereinhibitory to E. coli, though the 1:32 dilution was not. Repetition ofthis assay in the presence of supplemented KI and LP did not accentuatethe effects observed using H₂O₂ directly, with a 1:32 dilution still notinhibitory to the bacteria (FIG. 2), although the levels of H₂O₂ wouldhave been significantly reduced during the incubation, in this case.This is because the supplemented LP would use available peroxide tocatalyse the production of reactive oxygen species using thesupplemented KI as substrate.

The pattern of inhibition caused by the addition of the sodiumpercarbonate solution to inoculated broths (FIG. 3) was identical tothat observed following direct H₂O₂ addition. It is clear that there wasno difference as regards inhibition, following supplementation of KI andLP when using sodium percarbonate as the source of hydrogen peroxide(FIGS. 3 and 4) although the peroxide would have been used to producethe less toxic reactive oxygen species, hypoiodate, during theincubation.

It was clear that only a 1:2 and 1:4 dilution of the glucose/glucoseoxidase solution added produced sufficient H₂O₂ to be inhibitory (FIG.5), but not at dilutions of 1:8 (partial), 1:16 or 1:32. It was clear,however, that on repetition of the glucose/glucose oxidase assay in thepresence of the KI and LP (FIG. 6), inhibition of E. coli occurred at amuch greater dilution (up to and including a 1:32 dilution) thanachievable in the absence of added KI and LP (FIG. 5).

This result clearly indicates that the enzymatic production of hydrogenperoxide at levels greatly lower than those that are inhibitory tobacteria are sufficient to drive the production of inhibitoryconcentrations of antimicrobial reactive oxygen species. This offers amethod to deliver a therapeutic dose of the antimicrobial reactiveoxygen species without accumulation of potentially toxic H₂O₂.

Example 4

Volumes of milk (10 ml) were inoculated with E. coli and supplementedwith the lactoperoxidase, glucose oxidase, potassium iodide andBeta-galactosidase as a mechanism to generate the antimicrobialcomposition of the invention. Concentrations of 3.75 mg/L glucoseoxidase (˜200 units/mg), 12 mg/L lactoperoxidase (≥80 units/mg), 120mg/L potassium iodide/thiocyanate, and 400 ml/L Beta-galactosidase(≥2,600 units/ml) or greater were sufficient to observe antibacterialactivity. At the optimised concentrations these mixtures proved lethalto up to 10⁸ cells/ml of E. coli (subculture of the solution to freshagar plates resulted in no recoverable cells.). Concurrent negativecontrols (excluding each of the components) resulted in an increase ofbacterial numbers over the proceeding 24 h in the milk. The composition,generated in this manner, worked efficiently in the first 2 hours toeradicate bacteria.

A preparation was designed for use in the treatment of bovine mastitisby an intramammary infusion method. A field trial (6 cows, 6 quarters)was conducted wherein cows were treated on 4 occasions post milking withthe proposed bactericidal composition produced by an enzymatic system(in this case, using Beta-galactosidase milk activation as an object ofthe present invention). The preparation contained lactoperoxidase (4mg≥80 units/mg), glucose oxidase (2 mg, ˜200 units/mg),Beta-galactosidase (0.4 ml, ≥2,600 units/ml), and potassium iodide (150mg) per dose. Significant decreases were noted in somatic cell counts ofthe animals as a result of treatment, recorded between 5 and 30 daysafter treatment. Results from the trial are tabulated below (Table 2),farms C and D.

TABLE 2 Somatic Cell Counts of treated animals. Initial SCC After 30 %Cow # (in millions) 5 days days Reduction Farm C 1 8.9 1.46 84 2 11.580.36 97 3 2.32 0.647 72 Farm D 118 11.094 1.234 89 529 24.942 18.278 27207 22.835 4.214 82

An attempt was made to determine the effects of the milk pasteurisationprocess on the enzymatic components, which can be used to produce thereactive oxygen species of the composition of the present invention.Working concentrations of the enzymes were heated in 50 μl volumes to72° C. and held for 15, 30, 60, 300 or 600 seconds.

Appropriate concentrations of these aliquots were then transferred tomilk containing the necessary components of the system and ˜10⁸ cells/mlE. coli. Total viable counts were performed after 24 hrs incubation at37° C. This allowed the determination of whether the enzymes were stillactive after heating. Glucose oxidase demonstrated activity after 300seconds heating, as did lactoperoxidase. The activity ofBeta-galactosidase was, however, impaired by the heating process, with atypical pasteurisation cycle (72° C. for 15 seconds) completelyinactivating the enzyme. This would suggest that bacterial startercultures used in post-processing of milk (yogurt and cheese) would notbe inhibited by the use of the described preparations to generate thereactive oxygen species of the present invention.

A 10 ml volume of milk was used a model to test the hypothesis that achloroperoxidase enzyme could be used instead of lactoperoxidase togenerate antimicrobial reactive oxygen species. Milk was again spikedwith E. coli (˜10⁷ cells/ml). A suitable concentration of bothBeta-galactosidase (2-3 μl, ≥2,600 units/ml) and glucose oxidase (15 μlof a 2.5 mg/ml solution, ˜200 units/mg) was supplemented to the milk. Apreparation of chloroperoxidase (0.5 μl, ≥11,100 units/ml) was thenadded, and resulted in a complete eradication of E. coli cells within 24hours, at 37° C. Control experiments, without each, or two of theenzymes, resulted in the proliferation of a bacterial numbers within thesame incubation conditions (10⁸⁻⁹ cells/ml).

Example 5

A field trial was conducted using a supplemented glucose based enzymaticsystem to produce the reactive oxygen species and to assess the efficacyof such an approach to producing the antimicrobial agents. A number ofcows (8) were treated twice a day for two days with the supplied system.These cows demonstrated a marked decrease in their somatic cell counts(in the region of 50% after 5 days, see Table 3, Farms A and B, followedby a similar decrease after a further 5 days for Farm B, wheresubsequent data was available), a proxy method of measuring bacterialload. This demonstrated the efficacy of the lactoperoxidase system ingenerating the antimicrobial species, without use of Beta-galactosidase,to give assurance that it was a feasible source for generation of thebactericidal species.

TABLE 3 Somatic Cell Counts of treated animals using a glucosesupplementation system to produce the antimicrobial reactive oxygenspecies. Initial SCC After 10 % Cow # (in millions) 5 days daysReduction Farm A 438 3.016 1.68 42.3 852 6.981 3.476 50.2 892 1.56 0.51666.9 717 6.331 0.218 96.6 Farm B 794 3.4 0.926 0.59 82 501 0.71 0.470.394 44.5 831 3.5 3.3 0.995 71 823 0.812 0.554 0.186 77

Example 6

A trial was conducted to determine the ability of the proposedcomposition to eradicate biofilm-based bacterial cells. This wasperformed using two culturing techniques. A continuous culture of E.coli was established using a chemostat. This system is designed to allowthe operator the ability to control the growth rate of the organism.Most infections will occur as a result of slow growing cells (due tolimited nutrient availability). This phenotype will have an effect onthe tolerance of cells to antimicrobials, and is more realistic of thehost environment. Further to this, these cultures were used to growbiofilms on a Modified Robbins Device, wherein, the cells were allowedto attach and proliferate on the surface of a polyurethane coupon. Thesecells share the phenotype of biofilm cells noted in a typical infectionin mastitis, CF/TB lungs, wounds, burns, and on medical devices andrespond in a similar fashion and offer a viable model for antimicrobialtesting.

The antimicrobial species were produced on test coupons using thelactoperoxidase based system, containing lactoperoxidase (2 mg/L, ≥80Units/mg), potassium iodide, (300 mg/L), glucose (12.5 g/L) and glucoseoxidase (0.57 mg/L, ≥200 Units/mg), by submerging the coupon in thesolution at 37° C. for 24-48 hours. Control coupons were also treatedwith a saline solution or a mixture of the system lacking in one of thecomponents.

Cells were then recovered from the surface of the coupons by means ofsonication, and their viability determined. Cells treated with salineonly were viable (10⁵ cells/coupon), as were those tested with thesystem lacking in one of the components required to produce theantimicrobial species. No viable cells were recovered from the couponswherein cells were exposed to the antimicrobial species produced by afully functioning LP system. This result compares favourably withsimilar previously reported treatment regimes using a variety ofantibiotics (“Linezolid compared with eperezolid, vanocmycin, andgentamicin in an in vitro model of antimicrobial lock therapy forStaphycoccus epidermidis central venous catheter-related biofilminfections”, Curtin et al., 2003, Antimicrobial Agents and Chemotherapy,Vol. 47, No. 10, p 3145-3148), wherein cells were still recoverableafter 10 days with treatment of 10 g/L gentamycin and 7 days withtreatment of 10 g/L vancomycin, though better results were recorded forlinezolid and eperezolid. Such concentrations are far greater (up to1,000 fold) than those that would be lethal to the same strain grownplanktonically. By contrast, the concentrations of the antimicrobialspecies generated in the present experiment were of the same order ofmagnitude as those used to kill planktonic cells.

Example 7

The susceptibility of a number of P. aeruginosa strains to theKI-Dose-150 composition was tested using the method described inExample 1. These strains were of interest as they demonstrated increasedtolerance to a variety of key antibiotics typically used to treat lunginfections associated with cystic fibrosis, and were recovered from thesputum of cystic fibrosis patients presenting with lung infection. Therelative susceptibilities are described in Table 4. As evident fromTable 4, the antibiotic tolerant strains of P. aeruginosa are no moretolerant to KI-Dose-150, indicating that the system would be effectiveat treating such infections when delivered to the lung. ‘S’ denotessensitive, ‘R’ denotes resistant or increased tolerance.

TABLE 4 Susceptibility of antibiotic tolerant P. aeruginosa strains to‘KI-Dose-150’. Amikacin Tobramycin Ciprofloxacin Gentamicin MIC PA01(wild type) s s s s 0.25 mM R550/2012 9026 R R R R 0.12 mM R468/20129027 R s s s 0.06 mM R479/2012 9028 R R s R 0.12 mM R480/2012 9029 R s RR 0.12 mM P. aeruginosa s s R s 0.25 mM D12 The MIC value represents theminimum level of reactive oxygen species (hypoiodate) required to killthe strains (millimoles per litre produced over 24 hours).

Example 8

The treatment of respiratory infections by antibiotics will typically bedelivered using oral or intra venous drugs. The aerosolised form of theantibiotics can also be used to counteract the poor transfer of drugfrom blood across the alveoli of the lung to infection sites. Variousembodiments of the proposed antimicrobial composition were aerosolisedusing an AeroNeb nebuliser (courtesy of Aerogen Ltd.). Solutions ofhydrogen peroxide/glucose/glucose oxidase/lactoerpoxidase/iodide/orthiocyanate were passed through the nebuliser one at a time, or mixedtogether and passed through the nebuliser, and the aerosol collected ina sterile 25 ml container. The antimicrobial potency of the aerosolisedforms was compared to un-aerosolised forms (as described in Example 1),using doubling dilutions on a multi-well plate based assay. The proposedcomposition did not show any decreased activity when compared to thesolutions that were not aerosolised. In addition, it was demonstratedthat the enzymatic system, which can be used to produce theantimicrobial species was not affected by nebulisation. Specifically,the solutions did not show any reduced enzyme activity, or decreasedlevels of compound present when compared to stock solutions of the samecomponents. This model would suggest that the proposed antimicrobialsystem would be a good target or candidate for successful aerosoldelivery to the lung to treat respiratory infections.

Example 9

Lactoferrin is a mammalian protein that has been characterised toexercise antimicrobial properties, particularly on biofilm. As such, itis a good potential compound that could target and disrupt biofilmproduction in an infection model, and one that could act synergisticallywith a system such as the antimicrobial composition of the invention.The relative antimicrobial potencies of KI-Dose-150 and Thio-Dose-150were tested as described in Example 1, both in the presence and absenceof varying concentrations of lactoferrin. The presence of supplementedlactoferrin to the thiocyante model did not enhance the antimicrobialproperties already present (ratios of lactoferrin to thiocyanate were1:1, 1:2, 1:4). This would suggest that the presence of lactoferrin atsignificant concentrations does not inhibit the actions of alactoperoxidase-thiocyanate model to produce the antimicrobial species.Planktonic cells were used, allowing the possibility that an accentuatedantimicrobial effect would be noted for treatment of an actualinfection.

The same ratios for lactoferrin to iodide did, however, lead to a notedtwo-fold increase in antimicrobial activity of thelactoperoxidase-iodide model for production of the antimicrobialspecies, suggesting that it would be a suitable companion in a proposedtreatment regime. In the absence of lactoferrin, a 256-fold dilution ofthe system was still inhibitory to E. coli. With the addition oflactoferrin (at the three described ratios), a dilution factor of 512was also inhibitory to the bacterial culture. Infection sites will oftenbe composed of biofilm, which would allow an increased activity of thelactoferrin to be noted.

Example 10

Antibiotic therapies designed for the treatment of bovine mastitis willoften induce an inflammation of the udder, leading to an increase in thesomatic cell count for the animal. This is disadvantageous in that theprice of the sold milk depends on a low somatic cell count. A number ofdrugs can typically be added to counter the inflammation arising due tothe intramammary infusion of an antimicrobial therapy. Prednisone (orits active form, prednisolone) is a glucocorticoid steroid used tominimise an undesired immune response. A dosage of 10-20 mg is oftenadministered in conjunction with antibiotic based intramammary infusionsto halt an increase in the somatic cell count. An in vitro experimentusing a dose of the proposed lactoperoxidase system (KI-Dose-150) in theabsence and presence of either predisone or prednisolone did not resultin any decrease in antimicrobial potency of the composition. This wouldindicate that the use of a typical dose of either drug would notinterfere with the ability of the composition of the invention toeradicate bacteria in the udder (or other environment), and would helpminimise increase of the somatic cell count.

Example 11

The ability of an enzymatic system to produce the antimicrobial specieson a continuous basis was determined by repeat inoculations of bacterialculture to an antibacterial component containing solution. A 10 mlvolume of LB growth medium was supplemented with glucose oxidase (15 μlof 2.5 mg/ml, 200 Units/mg), beta-galactosidase (30 μl, 10 mg/ml, 48,000Units/mg), lactoperoxidase (20 μl mg/ml, 80 Units/mg) potassium iodide(30 μl, 40 mg/ml), with a final concentration of 2% lactose.Approximately 10⁸ cfu of E. coli ATCC 25922 were added and the mixturewas incubated overnight, at 37° C. After 24 hours, no cells wererecoverable to a fresh nutrient agar plate. A further inoculum ofapproximately 10⁸ cfu of E. coli ATCC 25922 cells was then added to thevolume and the mixture was again allowed to incubate overnight. Similar,subsequent inoculations (ten inoculations, at one day intervals) of thebroth with the bacterium did not result in growth or the recovery ofbacterial cells. This result demonstrates that the concentration of theantimicrobial reactive species was held sufficiently above bactericidallevels over a significant time period.

Example 12

The effect of substrate choice on the potency of the compositionproduced by various systems was determined using variations of aninhibition growth assay, firstly, where the concentration of H₂O₂(produced by the enzymatic reaction of glucose and glucose oxidase) wasconstant and the concentration of the chosen substrates was altered.Secondly, an assay where the concentration of substrates was maintainedconstant, and the H₂O₂ levels were varied was employed.

(i) Constant H₂O₂

E. coli (50 μl of an overnight culture) was added to Mueller Hintonbroth containing 5 μl glucose oxidase (2.5 mg/ml) and 10 μl LP (4mg/ml). This was aliquoted (150 μl) to rows of a 96-well plate. Equalvolumes (150 μl) of either potassium iodide or potassium thiocyanate(both 40 mg/ml) were added to the initial well. The samples were doublydiluted as far as well 11, leaving well 12 as a control. The plate wasincubated overnight at 37° C., and the optical density measuredthroughout.

Result: Thiocyanate concentrations in dilutions 1 to 9 were at a levelsufficient for complete inhibition of the bacteria. Iodideconcentrations in dilutions 1 to 8 were at a level sufficient forcomplete inhibition of the bacteria. This result would initiallyindicate that there was no significant difference in the potency of thereactive species produced by either substrate, and whatever differenceswere noted could have been as a result of the difference in molarity ofthe concentrations.

(II) Constant Substrate E. coli (200 μl of an overnight culture) wasadded to 20 ml Mueller Hinton broth already containing 40 μl LP (4mg/ml) and 60 μl of either potassium iodide or potassium thiocyanate (40mg/me. This was aliquoted (150 μl) to a 96 well plate. Samples of a H₂O₂producing system (2 ml 20% glucose containing 10 μl glucose oxidase, 2.5mg/ml) were added to the initial well, and doubly diluted, leaving well12 as a control.

Result: Using a broth with a constant thiocyanate concentration, therewas inhibition in the two highest dilutions of sample (H₂O₂). However,on using iodide, there was inhibition of bacterial growth up to, andincluding, six dilutions of sample. This results indicates that there isa significant difference in the potency of the antimicrobial speciesproduced using an enzymatic system, which releases lower levels ofhydrogen peroxide, when the substrate level is maintained at a constantlevel. This would imply that there would be a distinct advantage inusing iodide when lower levels of H₂O₂ production are typical.Differences in molarity cannot explain the apparent difference inoutcome when using iodide instead of thiocyanate.

Repetition of (i) and (ii) using milk as the growth medium showed verysimilar results and patterns. Using an alternative source of H₂O₂(direct addition in this case) did not show up the peculiar outcomedifference between iodide and thiocyanate.

Example 13

A protocol was drawn up that allows an operator to determine theappropriate concentrations of the antimicrobial species of the inventionfor use in the inhibition/killing of bacterial cells in broth. Theprotocol is based on doubling dilutions of the components, similar tothat used in 96-well plate described in Example 1. A bacterial culture(10⁷ cfu/ml) is established and aliquoted to tubes (5 ml volumes addedto 15 ml tubes would allow sufficient headspace for required oxygen,with the first tube to contain double the volume, 10 mls. The brothshould contain sufficient appropriate sugar if using enzymes to produceH₂O₂, for example, glucose if using glucose oxidase, with 1-2% beingtypically sufficient).

An aliquot (preferably no more than 5% of the total volume of thesolution, 500 μl) of the hydrogen peroxide producing components areadded to the initial volume, and doubly diluted. After 24 hours thelevel of innate tolerance for a bacterial strain to hydrogen peroxidecan be determined by the pattern of growth/no growth in the tubes. Thehydrogen peroxide producing components could consist of a monosaccharideor disaccharide sugar and their appropriate cleaving enzymes (notablylactose and beta-galactosidase and glucose oxidase, or glucose andglucose oxidase) or more simply hydrogen peroxide can be added directly,or as hydrogen peroxide releasing percarbonate or citric acid, etc.

In conjunction with the test to determine the innate hydrogen peroxidesensitivity of the test strain, the same test would also be used using areactive oxygen species producing solution (such as Thio-Dose-150 orKI-Dose-150, as described in example 1), wherein the hydrogen peroxideproducing components are present, as well as the peroxidise enzyme(chloroperoxidase or lactoperoxidase, and their appropriate substrates)are also present.

Although various ratios and concentration variations of the LP system,for example, can be employed to yield antimicrobial and bactericidalconcentration of the reactive species, the authors recommend a possibleratio of 75:2 of substrate to lactoperoxidase (for example, 150 mg KIand 4 mg lactoerpoxidase (at least 80 Units/mg). Similarly, if H₂O₂ isto be produced by the enzymatic cleavage of glucose, for example, theauthors recommend a 75:2:1 of substrate, lactoperoxidase, and glucoseoxidase (200 Units/mg glucose oxidase). The samples should be incubatedat the appropriate temperature, and shaken overnight. Control cultures(without, for example, LP components or H₂O₂) should yield bacterialgrowth. At sufficiently high concentrations of H₂O₂ (initial few tubes),growth should not be observed but as the H₂O₂ level drops (more dilutecultures), growth will be evident. This will allow the operator todetermine the innate tolerance of the strain to the actions of H₂O₂.Often, Streptococci strains are very susceptible to the actions of H₂O₂,as they lack catalase required to cleave the H₂O₂ molecule, whilst otherspecies will be tolerant of H₂O₂ at levels of ˜2 mM (some yeasts, forexample). The addition of the substrate and lactoperoxidase to theinitial tube should result in no growth at levels of H₂O₂ that were notpreviously inhibitory as the more potent reactive species are produced.Similarly, there will be a level at which the dilution was such thatgrowth occurred. The difference in outcome between ‘H₂O₂ only’ and H₂O₂and substrate and LP′ will inform the operator as to concentration ofthe LP-system components necessary to kill the test strain, or toinhibit its growth over 24 or 48 hours, as required. The authorsrecommend choosing a concentration at which the H₂O₂ is not inhibitoryin itself. Data described in Example 3 describes the use of an enzymaticcleavage of sugar, which allows a ‘window’ where the H₂O₂ levelsproduced in the solution are insufficient to cause inhibition, thoughsufficient to drive the production of the antimicrobial reactive oxygenspecies by the LP reaction (in instances where the strain does notproduce catalase, and is therefore extremely susceptible to the actionsof H₂O₂, the authors recommend using levels sufficient to kill typicalcatalase producing strains). A sub-culture to appropriate agar plate at24/48/72 hour time points will allow the operator to determine at whichconcentration the components are produced at bactericidalconcentrations, as opposed to bacteriostatic levels. The compositionwill be determined as bacteriocidal when no more than 0.001% of thestarting cell numbers are recoverable from the broth.

This test allows the operator to determine the bacteriocidalconcentration of the dose, and also the concentrations of hydrogenperoxide required to produce the components using an enzymatic system,without generating inhibitory levels of hydrogen peroxide. Suchinformation will be valuable if contemplating introducing an enzymaticsystem to a sensitive environment, such as the mammalian lung.

Existing statistical models will allow the operator to then ‘scale up’appropriately to determine the necessary levels required to treatinfections or large volumes of liquid etc, for example, the udder orlung.

Example 14

The lower limits of each of the components in an enzymatic system,required to produce inhibitory biocidal concentrations of theantimicrobial agents, were determined (for example Table 1 and Table 5).To establish these lower limits for each component, minimum inhibitoryconcentrations for each were calculated using doubling dilutions on a96-well plate, in a manner similar to that described in Example 1,wherein the concentration of the component of choice is lowered until noeffect on growth is noted. In 10 ml LB broth growth medium (with 2%glucose), replete with 120 mg/L KI, 320 units LP, at least 0.5 unitglucose oxidase/ml is required to produce hydrogen peroxide.

Concentrations below this resulted in insufficient hydrogen peroxidebeing produced to provide for the further production of the reactiveoxygen species at a bactericidal concentration. Similarly, the reductionof glucose levels requires an increase of glucose oxidase levels tocompensate; 1% glucose required 1 units/ml, while 0.5% glucose required2 units/ml activity glucose oxidase. In solutions where the glucoselevels and glucose oxidase levels are sufficient, the level of requirediodide (or thiocyanate) substrate was approximately 0.5 mM. At levelsbelow this, there was insufficient reactive oxygen species produced toresult in effective bactericidal activity. The level of LP required fora reaction to produce the reactive oxygen species at the requiredconcentrations was determined at 0.15 unit activity/ml (1 mM KIpresent). Levels below this resulted in little antibacterial activity.This embodiment of the invention suitable for the therapeutic treatmentof mastitis also included beta-galactosidase to convert the lactosepresent in milk to glucose. An in vitro examination of the requiredlevel of this enzyme was performed in milk (5% lactose), with 1 mM KI,0.75 units/ml glucose oxidase activity/ml (levels below this willproduce a ‘bottleneck’ in the enzymatic pathway resulting ininsufficient reactive oxygen species being produced), and 1 unitlactoperoxidase activity/ml present The required activity ofbeta-galactosidase lay at approximately 1.5 units activity/ml.Beta-galactosidase activity at levels below this did not result in theinhibition of bacterial growth or in killing of bacterial cells, butrather in bacterial proliferation.

Example 15

It is possible to produce the antimicrobial reactive oxygen species ofthe composition of the invention before adding it to the site ofinfections. This may be achieved by the mixing of the required enzymaticcomponents ensuring that the resulting reactive oxygen species (ROS;hypothiocyanate, hypoiodate, or hypochlorate) is produced outside of thetreatment site. Further to this, any excess hydrogen peroxide left afterthe reaction can be removed by the addition of catalase (which reactswith hydrogen peroxide, producing oxygen gas and water). This may provea very safe method of delivering the chosen ROS without the potentiallydisadvantageous hydrogen peroxide molecules.

Similarly, it is possible to introduce the catalase at the infectionsite also to help ‘quench’ the potential build-up of harmful hydrogenperoxide.

To demonstrate this, the potency of the KI-Dose-150 and Thio-Dose-150compositions were tested, using the protocol described in Example 1, ina broth growth medium containing catalase (20 μl of a 4 mg/ml, >1,000units/mg to 20 ml broth). The potency of the doses was not reversed. A1:1024 dilution of the doses was inhibitory in the absence of catalase,and a 1:512 dilution of the doses was inhibitory in the presence ofcatalase.

As a comparison, the test was performed using only hydrogen peroxide(0.85 M), both in the presence and absence of catalase. The catalaselevel was sufficient to completely reverse the inhibitory nature ofhydrogen peroxide, indicating that the catalase levels used for theexperiment were sufficient to ‘quench’ the activity, and thus, would beappropriate component to ‘mop up’ excess hydrogen peroxide produced ifthe iodide or thiocyanate substrate is used up, but not inhibit thereaction per se when substrate is still present.

This may serve to protect mammalian tissue.

Example 16

A further example of a pre-activated system was employed as follows:solutions (4 ml volumes) containing 0.85 M H₂O₂ plus none or 2.5 MNaCl/5 μl chloroperoxidase (˜10,000 Units/ml) were allowed incubate. Thesolutions were then split and either catalase treated (50 μl of a 4mg/ml, >1,000 units/mg) or were not catalase treated. The relativeantimicrobial properties of the solutions were then tested using theprotocol as described in Example 1 using E. coli supplemented broth.Inhibition of the bacteria was noted at >1:640 dilutions for hydrogenperoxide only, hydrogen peroxide+chloroperoxidase/NaCl, and the catalasetreated hydrogen peroxide+chloroperoxidase/NaCl samples. However, therewas no inhibition noted for the catalase treated hydrogen peroxidesample. This result would suggest that it is possible to remove anyexcess hydrogen peroxide by means of catalase treatment, withoutreducing the potency of the reactive oxygen species. The solutions wereallowed to incubate for longer, after which time (72 hours) the resultwas repeated. This would suggest that this form of the ROS wasrelatively stable and could be prepared in advance of use.

Example 17

The protocol described in Example 1 was used to test ‘KI-Dose-150’, aversion lacking iodide and lactoperoxidase, as well as version lackingglucose oxidase. All three were tested against Candida glabrata, Candidakrusei, Candida tropicalis, Candida albicans and Saccharomycescerevesiae. Protocols were carried out for the Candida strains andSaccharomyces strain in nutrient broth and LB broth respectively, eachsupplemented with 2% glucose using the method described in Example 1.Results are presented in Table 5. It is clear from Table 5 that allstrains are inhibited by the actions of ‘KI-Dose-150’ and that thereactive oxygen species are thus antimicrobial and not justantibacterial. The levels of hydrogen peroxide produced in thesedilutions of the composition were themselves non-inhibitory to thetested strains.

TABLE 5 Susceptibility of fungal and yeast strains to ‘KI-Dose-150’. MICCandida albicans 0.25-0.5 mM Candida tropicalis 0.12-0.25 mM Candidaglabrata 0.25-0.5 mM Candida krusei 0.25-0.5 mM Saccharomyces cerevisiae0.12-0.25 mM The MIC value represents the minimum level of reactiveoxygen species (hypoiodate) required to kill the strains (millimoles perlitre produced over 24 hours)

Example 18

The results presented in Example 3 demonstrate that there are threecrucial levels of H₂O₂. These levels can be described using a schematicmodel, as illustrated in FIG. 7. Firstly, there is a higher thresholdlevel of H₂O₂, at or above which inhibition of bacterial growth occursas a direct result of the concentration of H₂O₂ in the growth medium.This is not the preferable mechanism of action for an antimicrobialcomposition, as H₂O₂ is toxic to host cells, and has been linked tomammalian tissue damage, for example.

The second threshold level of H₂O₂ is that required for the effectiveproduction of the antimicrobial reactive oxygen species. The experimentspresented herein (Example 3) describe the distinct advantage that isconferred by the use of an enzymatic method of H₂O₂ production, whereinthe levels of H₂O₂ can be maintained within this required ‘window’ (FIG.7) for a longer period of time (i.e. these are concentrations of H₂O₂that are effective at allowing the production of the requiredconcentration of reactive oxygen species, but that are not toxic inthemselves).

Lastly, the third threshold level of H₂O₂ is one at which there isinsufficient H₂O₂ to inhibit or provide for the production of thereactive oxygen species using an enzymatic system.

Using a more direct source of peroxide (such as the sodium percarbonateor hydrogen peroxide itself) results in a high initial concentration ofH₂O₂ that quickly decreases to a level that is ineffective for theproduction of the desired reactive oxygen species (FIG. 7).

Example 19

The conversion of substrate to the antimicrobial reactive oxygen species(ROS) was estimated by direct measurement of the relevant substrateconcentration during conversion and, for example, after 24 hours. Thevarious antimicrobial reactive oxygen species are relativelyshort-lived, but have variable half-lives depending on the substrateused, so a direct titration was not useful. For example, Thiocyanateconcentrations, at 1× (1.36 mM) and 5× (6.8 mM) levels, were comparedbefore and after incubation in a solution containing glucose,lactoperoxidase, and glucose oxidase. These were compared to a standardconcentration curve of thiocyanate levels (0, 0.0625, 0.125, 0.25, 0.5,1, 2, and 5× concentrations) using a colourometric assay as follows:

Five grams ferric chloride was suspended in 50 ml water. Any undissolvedferric chloride was removed by centrifugation, leaving ˜30 ml ferricchloride solution. To cuvettes, 150 μl of the ferric solution was added,followed by the addition of 700 μl water. A volume of 10× thiocyanatewas added to each cuvette (200 μl, 160 μl, 100 μl, 40 μl, 20 μl, 10 μl,5 μL, 25 μl (1:10), 12.5 μl (1:10), 0 μl). The final volume in thecuvette was brought to 1,050 μl by the addition of water. For thesample, 50 μl of the previously incubated 1× or 5× dilutions were added,plus 150 μl water. The optical density was recorded at 460 nm, and theconcentrations were then calculated using a standard curve. Theresulting standard curve had an r2 value of >0.99, (see FIG. 8)indicating that it was an accurate method of determining an unknownconcentration of thiocyanate.

The 1× dose (left incubating overnight with the enzyme system), read as0.17× dose after 24 hours. Similarly, the 5× dose (left incubatingovernight with the enzyme system), read as a 1.1× dose after 24 hours.Both of these results would indicate that, under these conditions, therewas an 80-85% drop in thiocyanate levels. Assuming a 1:1 ratio ofthiocyante loss to ROS production (in this case, OSCN-), this allows thedetermination of the ROS levels produced to be in the region of 1 mM,and 5 mM over the 24 hours for the 1× and 5× doses, respectively. Thisvalue can be adapted using a higher substrate concentration, whilstmaintaining efficiency in conversion, at least within the range testedhere.

The specific levels of ROS disclosed here as providing bactericidal andfungicidal activity are greater than the levels produced using the LPsystem elsewhere. The concentration dependent microcidal effectdescribed in this application; and the ability to determine minimuminhibitory concentrations for the ROS against target strains and invarious media and settings, allows the use of the composition of theinvention as a targeted bacteriocidal and microcidal therapeutic andantimicrobial composition, as opposed to applications with merelygeneral non-specific bacteriostatic effects.

Example 20

The ability to achieve potentially therapeutic doses of the reactiveoxygen species in vivo was investigated, again using a milk model. Theintramammary infusion method was used to introduce the describedprotoype of Example 4, [150 mg KI, 4 mg lactoperoxidase (320 units), 2mg glucose oxidase (400 units), and Beta-galactosidase, (1,350 Units)]to a bovine udder. This was performed after milking of the animal. Atthe next milking, a sample of milk was obtained. Aliquots (10 mlvolumes) of the milk were spiked with approximately 10⁷ cfu/ml ofbacterial strains (E. coli, P. aeruginosa, or S. dysgalactiae) andallowed incubate overnight at 37° C. whilst shaking. A total viablecount was performed using agar plates. The milk was completelyinhibitory to the strains. This would indicate the presence of thereactive oxygen species in the milk at a concentration sufficient tokill these mastitis causing organisms. This is important indemonstrating the technology as a therapeutic, Further to this, becausethe reactive oxygen species are relatively short-lived, it is likelythat the concentration would have been higher in the udder itself,increasing the effectiveness of the treatment further.

Compositions Suitable for Administration.

A solution containing containing 1-100,000 Units activity glucoseoxidase, 1-100,000 Units activity of lactoperoxidase, and 0.1-10,000 mgthiocyanate/iodide, and 0.01-100,000 Units activity ofbeta-galactosidase would be suitable to be administered to the udder ofan animal as an intramammary infusion

A solution containing containing 1-100,000 Units activity galactoseoxidase, 1-100,000 Units activity of lactoperoxidase, and 0.1-10,000 mgthiocyanate/iodide, and 0.01-100,000 Units activity ofbeta-galactosidase would be suitable to be administered to the udder ofan animal as an intramammary infusion

A solution containing containing 1-100,000 Units activity glucoseoxidase, 1-100,000 Units activity of lactoperoxidase, and 0.1-10,000 mgthiocyanate/iodide, and 0.01-100,000 mg glucose would be suitable to beadministered to the udder of an animal as an intramammary infusion

A solution containing containing 1-100,000 Units activity glucoseoxidase, 1-100,000 Units activity of lactoperoxidase, and 0.1-10,000 mgthiocyanate/iodide, and 0.01-100,000 mg glucose would be suitable to beadministered to the lungs for the treatment of bacterial infection as anebulised spray.

The same solution as above containing supplemented lactoferrin(0.01-100,000 mg), prednisone (0.01-100,000 mg), or prednisolone(0.01-100,000 mg), catalase (1-1,000,000 Units) or a combination of twoor more of these.

A solution containing containing 1-100,000 Units activity glucoseoxidase, 1-100,000 Units activity of chloroperoxidase, and 0.1-10,000 mgchloride ion, and 0.01-100,000 mg glucose to be administered to thelungs for the treatment of bacterial infection as a nebulised spray.

The same solution as above containing supplemented lactoferrin(0.01-100,000 mg), prednisone (0.01-100,000 mg), or prednisolone(0.01-100,000 mg), or a combination of two or more of these. A solutioncontaining containing 1-100,000 Units activity of lactoperoxidase, and0.1-10,000 mg thiocyanate/iodide, and 0.01-100 ml hydrogen peroxidewould be suitable to be administered to the lungs for the treatment ofbacterial infection as a nebulised spray.

A poultice impregnated with 1-100,000 Units activity glucose oxidase,1-100,000 Units activity of lactoperoxidase and an accompanying gelcontaining 0.1-10,000 mg thiocyanate/iodide, and 0.01-100,000 mg glucoseto be applied to the poultice prior to its use in treating burns or openwounds of a patient.

A poultice impregnated with 1-100,000 Units activity glucose oxidase,1-100,000 Units activity of chloroperoxidase and an accompanying gelcontaining 0.1-10,000 mg chloride ion, and 0.01-100,000 mg glucose to beapplied to the poultice prior to its use in treating burns or openwounds of a patient.

The same poultices as above containing supplemented lactoferrin(0.01-100,000 mg), prednisone (0.01-100,000 mg), or prednisolone(0.01-100,000 mg), catalase (1-1,000,000 Units) or a combination of twoor more of these. A variety of medical devices may be coated/impregnatedwith 1-100,000 Units activity glucose oxidase, 1-100,000 Units activityof lactoperoxidase before insertion into the body of a patient.

A pre-prepared composition containing iodide/thiocyanate ions(0.1-10,000 mg) allowed to react fully with hydrogen peroxide (0.01-100ml) in the presence of lactoperoxidase (0.01-1,000,000 Units). Thecomposition is catalase treated (0.01-1,000,000 Units) to remove excesshydrogen peroxide, before the composition is used to treat infectionsites.

A pre-prepared composition containing chloride ions (0.1-10,000 mg)allowed to react fully with hydrogen peroxide (0.01-100 ml) in thepresence of chloroperoxidase (0.01-1,000,000 Units). The composition iscatalase treated (0.01-1,000,000 Units) to remove excess hydrogenperoxide, before the composition is used to treat infection sites.

A pre-prepared composition containing chloride ions (0.1-10,000 mg)allowed to react fully with sodium percarbonate (0.01-100,000 mg) in thepresence of chloroperoxidase (0.01-1,000,000 Units). The composition iscatalase treated (0.01-1,000,000 Units) to remove excess hydrogenperoxide, before the composition is used to treat infection sites.

A pre-prepared composition containing thiocyanate/iodide ions(0.1-10,000 mg) allowed to react fully with sodium percarbonate(0.01-100,000 mg) in the presence of lactoperoxidase (0.01-1,000,000Units). The composition is catalase treated (0.01-1,000,000 Units) toremove excess hydrogen peroxide, before the composition is used to treatinfection sites.

A pre-prepared composition containing chloride ions (0.1-10,000 mg)allowed to react fully with glucose (0.01-100,000 mg) and glucoseoxidase (0.1-1,000,000 Units) in the presence of chloroperoxidase(0.01-1,000,000 Units). The composition is catalase treated(0.01-1,000,000 Units) to remove excess hydrogen peroxide, before thecomposition is used to treat infection sites.

A pre-prepared composition containing thiocyanate/iodide ions(0.1-10,000 mg) allowed to react fully with glucose (0.01-100,000 mg)and glucose oxidase (0.1-1,000,000 Units) in the presence oflactoperoxidase (0.01-1,000,000 Units). The composition is catalasetreated (0.01-1,000,000 Units) to remove excess hydrogen peroxide,before the composition is used to treat infection sites.

The above pre-prepared solutions supplemented with lactoferrin (0.001mg-10,000 mg), prednisone (0.001 mg-10,000 mg), or prednisolone (0.001mg-10,000 mg), either individually or a combination of two or more.

The words “comprises/comprising” and the words “having/including” whenused herein with reference to the present invention are used to specifythe presence of stated features, integers, steps or components but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

1. A microbiocidal composition comprising a reactive oxygen species orcomponents capable of producing a reactive species, the compositionbeing capable of delivering the reactive oxygen species to a level of atleast 0.4 millimoles per litre, over a 24 hour period.
 2. A compositionas claimed in claim 1 capable of delivering the reactive oxygen speciesto a level of at least 0.5 millimoles per litre, over a 24 hour period.3. A composition as claimed in claim 1 or 2 wherein the reactive oxygenspecies is produced by the reaction of a peroxidase, a substrate for theperoxidase and hydrogen peroxide.
 4. A composition as claimed in anypreceding claim where the reactive oxygen species is selected from thegroup comprising hypothiocyanate (hypothiocyanite, SCNO⁻), hypoiodate(IO⁻) and hypochlorite (CLO⁻).
 5. A composition as claimed in claim 3 or4 wherein the peroxidase enzyme is selected from the group comprising alactoperoxidase, a chloroperoxidase, a bromoperoxidase and aniodooxidase.
 6. A composition as claimed in claim 5 comprising alactoperoxidase enzyme and further comprising iodide or thiocyanateions.
 7. A composition as claimed in claim 5 comprising achloroperoxidase enzyme and further comprising chloride ion.
 8. Acomposition as claimed in any of claims 3 to 7 where the source ofhydrogen peroxide is a solution of the hydrogen peroxide.
 9. Acomposition as claimed in any preceding claim where the hydrogenperoxide is released by a hydrogen peroxide releasing compound selectedfrom the group comprising percarbonates, citric acid and perhydrates, orby enzymatic methods.
 10. A composition as claimed in any of claims 3 to9 where the hydrogen peroxide is produced by an enzymatic reactionbetween a sugar and its appropriate oxidoreductase
 11. A composition asclaimed in claim 10 wherein the oxidoreductase is galactose oxidaseand/or glucose oxidase.
 12. A composition as claimed in claim 11 furthercomprising free monosaccharide sugar(s).
 13. A composition as claimed inany of claims 3 to 12 further comprising a disaccharide sugar, and itscorresponding glycoside hydrolase to produce a source of hydrogenperoxide.
 14. A composition as claimed in claim 13 wherein the glycosidehydrolase is Beta-galactosidase, and the disaccharide sugar is lactose.15. A composition as claimed in any preceding claim wherein a glycosidehydrolase and/or an oxidoreductase is used to react with sugars presentat the infection site.
 16. A composition as claimed in claim 10 whereinthe additional source of hydrogen peroxide is derived from the reactionof a polyol (sugar alcohol) with its relative oxidase enzyme.
 17. Acomposition as claimed in claim 16 wherein the polyol is glycerol andits relative oxidase enzyme is glycerol oxidase, or wherein the polyolis mannitol and its relative oxidase enzyme is mannitol oxidase.
 18. Acomposition as claimed in any of claims 2 to 17 wherein the hydrogenperoxide is produced from the enzymatic reaction of L-amino acids withL-amino acid oxidase or xanthine (or hypoxanthine) and xanthine oxidase.19. A composition as claimed in any preceding claim further comprisingeither hypoxanthine or xanthine or both.
 20. A composition as claimed inclaim 19 further comprising free amino acids.
 21. A composition claimedin any preceding claim additionally comprising lactoferrin, or aglucocorticoid.
 22. A composition claimed in claim 21 wherein theglucocorticoid is prednisolone or prednisone.
 23. A composition asclaimed in any preceding claim adapted for intramammary infusion ornebulisation, for use as an antimicrobial solution, emulsion or driedproduct, for use as an antimicrobial solution to be added to a poulticefor a burn lesion to the skin, for use as an antimicrobial solution tobe used as a nasal rinse, for use as an antimicrobial solution to beused as a surface cleaner.
 24. An intramammary infusion delivery deviceloaded with a composition as claimed in any preceding claim.
 25. Acomposition as claimed in any preceding claim where the solution isprepared as an emulsion, or dried product.
 26. A composition as claimedin any preceding claim wherein the compounds are adhered to the surfaceof a medical device.
 27. A composition as claimed in any preceedingclaim where the components are adapted for delivery sequentially orcumulatively to the infection site.
 28. A composition as claimed in anypreceeding claim where the components are allowed to react beforeaddition to the infection site.
 29. A composition as claimed in anypreceeding claim where the components are allowed react before additionto the infection site, and treated with catalase to remove excesshydrogen peroxide.